The present invention relates generally to the detection of microorganisms using peptides adapted for specific binding thereto and relates more particularly to a method for discovering such peptides.
Microorganisms, such as bacteria, viruses, fungi and protozoa, are commonplace in the environment. Although many such microorganisms are innocuous to humans, certain species of microorganisms are pathogenic and pose a serious health risk to people. Exposure to such pathogenic microorganisms may be inadvertent, such as in the case of poorly handled or poorly prepared foods containing Salmonella, E. coli O157:H7 or the like, or may be deliberate, such as in the case of biological weapons armed with spores of anthrax or the like. As can readily be appreciated, in view of the above, it is highly desirable to be able to detect the presence of pathogenic microorganisms in various media, such as food, water and air, that are likely to come into human contact. Unfortunately, the presence of pathogenic microorganisms in such media cannot typically be ascertained simply by visual or other sensory examination of the media, but rather, requires the use of specialized testing equipment and procedures. Moreover, because certain pathogenic microorganisms may be lethal in very small doses (for example, in some instances, in doses constituting as few as about ten microorganisms), comparatively large quantities of materials must often be tested for the presence of comparatively small quantities of pathogens.
One technique that is commonly used to detect the presence of a pathogenic microorganism in a sample is an enzyme linked immunosorbent assay (ELISA), such a technique employing, among other things, an antibody adapted to bind to the pathogen of interest. Examples of ELISA techniques used in the detection of pathogenic microorganisms may be found in the following U.S. patents: U.S. Pat. No.6,174,667, inventors Huchzermeier et al., which issued Jan. 16, 2001; U.S. Pat. No. 6,124,105, inventors Verschoor et al., which issued Sep. 26, 2000; U.S. Pat. No. 5,294,537, inventor Batt, which issued Mar. 15, 1994; and U.S. Pat. No. 4,486,530, inventors David et al., which issued Dec. 4, 1984.
Unfortunately, some difficulties that are commonly encountered in using ELISA technology to detect pathogens include the lack of long-term stability and/or insufficient specificity to bind only to the pathogenic microorganism of interest. Consequently, certain non-pathogenic microorganisms often become bound to such antibodies, thereby leading to the undesirable occurrence of false positive results.
Another technique that is commonly used to detect the presence of a pathogenic microorganism in a sample is a DNA-based approach that involves detecting within the sample the presence of one or more genes indicative of the pathogenic microorganism of interest. Such a DNA-based approach typically comprises inoculating a culture broth with a sample under investigation, allowing the broth to culture for a period of time (e.g., typically overnight up to a few days), isolating any microorganisms present in the broth, retrieving the DNA from the isolated microorganisms, amplifying the retrieved DNA, and using one or more hybridizing probes specific for a gene or genes of interest to detect the presence of said gene(s) within the amplified DNA. Although the aforementioned DNA-based approach does not typically suffer from the shortcoming of false positive results that are encountered in the above-described ELISA technique, it can readily be appreciated that the aforementioned DNA-based approach can be rather time-consuming, especially where there is a large quantity of sample to be tested.
Examples of other techniques for detecting the presence of a pathogenic microorganism in a sample are disclosed in U.S. Pat. No. 6,159,719, inventors Laine et al., which issued Dec. 12, 2000; U.S. Pat. No. 5,750,357, inventors Olstein et al., which issued May 12, 1998; Pyle et al., “Sensitive Detection of Escherichia coli O157:H7 in Food and Water by Immunomagnetic Separation and Solid-Phase Laser Cytometry,” Appl. Environ. Microbiol., 65(5): 1966–72 (May 1999) and Sharma et al., “Semi-automated fluorogenic PCR assays (TaqMan) for rapid detection of Escherichia coli O157:H7 and other Shiga toxigenic E. coli,” Molecular and Cellular Probes, 13:291–302 (1999). Unfortunately, many of the techniques described in the aforementioned publications suffer from one or more of the types of shortcomings described above.
In PCT Application No. PCT/US99/00771, inventor Tumbough, which was published Jul. 22, 1999, there is disclosed a technique for identifying peptides from a combinatorial library that bind specifically to bacterial spores of interest and that, therefore, can be used as capture probes or the like for use in detecting the presence of said spores in a sample. According to the aforementioned PCT application, said technique comprises mixing a quantity of a pathogenic spore with a commercially available (New England Biolabs) phage display library, said phage display library comprising a combinatorial library of random amino acid sequences (7-mer or 12-mer) fused to the minor coat protein (pIII) of the filamentous coliphage M13. After incubating for a sufficient period of time to allow the phage to complex with the pathogenic spores, the mixture is centrifuged to permit the isolation of the phage-spore complexes. The phage-spore complexes are washed repeatedly and the phage is then eluted from the phage-spore complexes with an elution buffer. The eluate is neutralized to prevent phage killing, and the eluted phage is then amplified by infecting E. coli. The cell lysate obtained from the culture is then subjected to the above series of steps repeatedly until about 3 to 4 rounds of biopanning take place. Next, individual clones are purified from the eluted phage, and phage plaques are amplified. The phage DNA is then extracted from each preparation to permit the DNA sequence that encodes the peptide to be determined.
One limitation to the above-described combinatorial phage display approach is that the peptide sequences being screened must be relatively short (i.e., in the range of 7 to 12 amino acid residues) to keep the number of members of the combinatorial library to a manageable size while, at the same time, assuring representation of all possible sequences within the library. Because of their comparatively short length, however, most of these peptides do not possess a stable secondary structure, thereby making binding specificity even more difficult.
Another limitation to the above-described combinatorial phage display approach is that it is restricted to identifying peptides from a combinatorial library that bind specifically to bacterial spores, as opposed to vegetative cells. As can readily be appreciated, because not all microorganisms form spores, an approach that is limited to spores excludes many microorganisms of interest. Moreover, as compared to spores, vegetative cells are actually active, there is clearly a considerable need to be able to detect vegetative cells as well as spores.